Laser scan confocal microscope

ABSTRACT

Fluorescence is generated from an irradiated point on an inspection surface of a sample and the fluorescence is collected by an objective lens. Here, because of the magnification chromatic aberration of the objective lens, the fluorescence going out from the objective lens travels along a path shifted from the irradiation light and changed substantially into a non-scan light by a galvano-scanner. The fluorescence passes through a dichroic mirror into a deflection system after light of unnecessary wavelength is removed by a filter. The deflection system is driven in synchronization with the galvano-scanner by a computer and corrects the shift and inclination of the optical axis generated by the magnification chromatic aberration of the objective lens. Then, the fluorescence forms an image of the irradiation point of the inspection surface of the sample on a pin hole of a pin hole plate by using a collective lens.

This is a Continuation of International Application No.PCT/JP2007/074511 filed Dec. 20, 2007, which claims the benefit ofJapanese Application No. 2006-345662 filed Dec. 22, 2006. The disclosureof the prior application is hereby incorporated by reference herein inits entirety.

TECHNICAL FIELD

The present invention relates to a laser scan confocal microscope.

BACKGROUND OF THE INVENTION

For example, in a confocal microscope disclosed in Japanese Patent No.3365884 (Patent Document 1), illumination light is collected onto asample such as a living sample, a light flux (fluorescent light) emittedfrom a focal portion on the sample is collected onto a confocaldiaphragm plane, and a light quantity of the light flux having passedthrough the confocal diaphragm is detected by a photodetector. In orderto obtain a two-dimensional image of the sample, the illumination lightis converted into scan light by a galvano-mirror type scanner or thelike, and the two-dimensional image of the sample is obtained while thesample is scanned with a focal portion (spot).

A pinhole member is disposed in the confocal diaphragm plane. Becausethe pinhole member transmits only a light beam collected in a pinhole(aperture) while cutting off other light beams, only the light beamemitted from a particular depth on the sample is incident to thephotodetector, and the light beams emitted from other depths are notincident to the photodetector. Therefore, in the confocal microscope, anobservation object can be sectioned only to an image of a thin-filmlayer located at the particular depth on the sample. In order to changesectioning resolution (thickness of a layer to be observed), it isnecessary to change an aperture diameter of the pinhole member. Thesectioning resolution is lowered when the aperture diameter isincreased, and the sectioning resolution is enhanced when the aperturediameter is decreased.

Because the sectioning resolution depends on an aperture diameter of thepinhole, it is convenient that the aperture diameter of the pinhole ischanged. Therefore, a method in which an aperture diameter of thepinhole can be changed like an iris diaphragm of a camera, a method inwhich plural pinholes having different aperture diameters are preparedsuch that one of the pinholes can be selected in a turret manner or asliding manner, and a method in which plural pinholes are prepared suchthat an optical path can be changed, are used.

Patent Document 1: Japanese Patent No. 3365884

Patent Document 2: Japanese Patent Application Laid-Open No. 2005-275199

However, when an aperture diameter of the pinhole is decreased toenhance the sectioning resolution, there is generated a limb darkeningphenomenon in which the light quantity is increased in the center of thescreen while decreased in a peripheral portion, the degree of whichdepends on the type of the objective lens. Therefore, even if the objecthas even brightness (intensity of generated fluorescent light), an imagehaving uneven brightness is obtained in the imaging result.

The limb darkening is mainly generated by vignetting and a shift of theconjugate point between a light source and the pinhole, caused bychromatic aberration of magnification. The conjugate point deviation hasthe following meaning. In fluorescent light observation an excitationwavelength differs from a fluorescent light wavelength. Accordingly,when the peripheral portion of the screen is scanned while the chromaticaberration of magnification exists in the optical system (particularly,in objective lens), the focal position of the fluorescent light is notmatched with the pinhole position due to the chromatic aberration ofmagnification, and the light quantity of the fluorescent light passingthrough the pinhole is decreased to darken the image of the peripheralportion.

In order to solve the problem of the limb darkening, for example, imageprocessing is performed to increase the light quantity of the peripheralportion in Japanese Patent Application Laid-Open No. 2005-275199 (PatentDocument 2). However, an optical correction is desired rather than theimage processing.

In view of the foregoing, an object of the invention is to provide alaser scan confocal microscope which can correct the influence of thelimb darkening caused by the chromatic aberration of magnification usingthe optical system even if the objective lens having the chromaticaberration of magnification is used.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the invention, a laser scanconfocal microscope includes a light source which is disposed at aposition conjugate to a specimen in an illumination optical system toemit illumination light; light separation means for separating theillumination light and observation light, the observation light beingemitted from the specimen; light scanning means for scanning theillumination light on the specimen; an objective lens which is disposedbetween the light scanning means and the sample to form a focal point onthe specimen; a collective lens which collects the observation light toa position conjugate to the focal point of the objective lens; a pinholeplate which is provided in a focal plane of the collective lens;detecting means for detecting observation light having passed through apinhole of the pinhole plate; and two-dimensional light deflection meanswhich is disposed between the light separation means and the pinholeplate, to be driven in synchronization with the light scanning means.

In accordance with a second aspect of the invention, in the laser scanconfocal microscope according to first aspect, a telescope opticalsystem is disposed between the light separation means and thetwo-dimensional light deflection means.

In accordance with a third aspect of the invention, a laser scanconfocal microscope includes a light source which is disposed at aposition conjugate to a specimen in an illumination optical system toemit illumination light; light separation means for separating theillumination light and observation light, the observation light beingemitted from the specimen; light scanning means for scanning theillumination light on the specimen; an objective lens which is disposedbetween the light scanning means and the sample to form a focal point onthe specimen; a collective lens which collects the observation light toa position conjugate to the focal point of the objective lens; a pinholeplate which is provided in a focal plane of the collective lens;detecting means for detecting observation light having passed through apinhole of the pinhole plate; and two-dimensional light deflection meanswhich is disposed between the light source and the light separationmeans, to be driven in synchronization with the light scanning means.

In accordance with a fourth aspect of the invention, in the confocalmicroscope according to the first or third aspect, the light scanningmeans and the two-dimensional light deflection means are agalvano-mirror type scanner.

In accordance with a fifth aspect of the invention, in the confocalmicroscope according to the first or third aspect, the two-dimensionallight deflection means corrects an optical path deviation between theillumination light and the observation light, the optical path deviationbeing generated according to a deflection amount of the light scanningmeans.

In accordance with a sixth aspect of the invention, a laser scanconfocal microscope includes a light source which is disposed at aposition conjugate to a specimen in an illumination optical system toemit illumination light; light separation means for separating theillumination light and observation light, the observation light beingemitted from the specimen; light scanning means for scanning theillumination light on the specimen; an objective lens which is disposedbetween the light scanning means and the sample to form a focal point onthe specimen; a collective lens which collects the observation light toa position conjugate to the focal point of the objective lens; a pinholeplate which is provided in a focal plane of the collective lens;detecting means for detecting observation light having passed through apinhole of the pinhole plate; and two-dimensional light deflection meansfor two-dimensionally shifting the pinhole plate, to be driven insynchronization with the light scanning means.

In accordance with a seventh aspect of the invention, in the confocalmicroscope according to the sixth aspect, the two-dimensionally shiftingmeans corrects an optical path deviation between the illumination lightand the observation light, the optical path deviation being generatedaccording to a deflection amount of the light scanning means.

In accordance with an eighth aspect of the invention, a laser scanconfocal microscope includes a light source which is disposed at aposition conjugate to a specimen in an illumination optical system toemit illumination light; light separation means for separating theillumination light and observation light, the observation light beingemitted from the specimen; light scanning means for scanning theillumination light on the specimen; an objective lens which is disposedbetween the light scanning means and the sample to form a focal point onthe specimen; a collective lens which collects the observation light toa position conjugate to the focal point of the objective lens; a pinholeplate which is provided in a focal plane of the collective lens;detecting means for detecting observation light having passed through apinhole of the pinhole plate; and a plane-parallel plate which isdisposed between an illumination lens and the light source, to be drivenin synchronization with the light scanning means, the illumination lensbeing provided between the light source and the light separation means.

In accordance with a ninth aspect of the invention, in the laser scanconfocal microscope according to the eighth aspect, two plane-parallelplates are provided while the two plane-parallel plates have rotatingaxes perpendicular to each other.

In accordance with a tenth aspect of the invention, in the confocalmicroscope according to the eighth aspect, the plane-parallel platecorrects an optical path deviation between the illumination light andthe observation light, the optical path deviation being generatedaccording to a deflection amount of the light scanning means.

In accordance with an eleventh aspect of the invention, the laser scanconfocal microscope according to any one of the first, the third, thesixth and the eighth aspects includes storage means in which arelationship between a deflection amount of the light scanning means anda drive amount of one of the two-dimensional light deflection means, themeans for two-dimensionally shifting the pinhole plate, the means fortwo-dimensionally shifting the collective lens, and the plane-parallelplate is stored for each objective lens used.

In accordance with a twelfth aspect of the invention, the laser scanconfocal microscope according to any one of the first, the third, thesixth and the eighth aspects includes storage means in which datarelating to chromatic aberration of magnification is stored for eachobjective lens used; and operation means for computing, using the data,a relationship between a deflection amount of the light scanning meansand a drive amount of one of the two-dimensional light deflection means,the means for two-dimensionally shifting the pinhole plate, the meansfor two-dimensionally shifting the collective lens, and theplane-parallel plate.

In accordance with a thirteenth aspect of the invention, the laser scanconfocal microscope according to the twelfth aspects includes correctionmeans for correcting the drive amount using scanning information on thelight scanning means and intensity information on the observation light.

In accordance with a fourteenth aspect of the invention, confocalmicroscope includes a light source which is disposed at a positionconjugate to a specimen in an illumination optical system to emitillumination light; light separation means for separating theillumination light and observation light, the observation light beingemitted from the specimen; light scanning means for scanning theillumination light on the specimen; an objective lens which is disposedbetween the light scanning means and the sample to form a focal point onthe specimen; a collective lens which collects the observation light toa position conjugate to the focal point of the objective lens; a pinholeplate which is provided in a focal plane of the collective lens;detecting means for detecting observation light having passed through apinhole of the pinhole plate; and correction means for correcting apositional relationship between a focal position of the observationlight and the pinhole, to be driven in synchronization with the lightscanning means.

Accordingly, the invention can provide the laser scan confocalmicroscope which can correct the influence of the limb darkening causedby the chromatic aberration of magnification using the optical systemeven if the objective lens having the chromatic aberration ofmagnification is used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing an outline of an optical system in a laser scanconfocal microscope according to a first embodiment of the invention.

FIG. 2 is a view showing an outline of an optical system in a laser scanconfocal microscope according to a second embodiment of the invention.

FIG. 3 is a view showing an outline of an optical system in a laser scanconfocal microscope according to a third embodiment of the invention.

FIG. 4 is a view showing an outline of an optical system in a laser scanconfocal microscope according to a fourth embodiment of the invention.

FIG. 5 is a view showing an outline of an optical system in a laser scanconfocal microscope according to a fifth embodiment of the invention.

FIG. 6 is a view showing a table used to determine a deflection amountby a first method.

FIG. 7 is a view showing a table used to determine the deflection amountby a modification of the first method.

FIG. 8 is a view showing a table used to determine the deflection amountby a second method.

DETAILED DESCRIPTION

Embodiments of the invention will be described with reference to thedrawings. FIG. 1 is a view showing an outline of an optical system in alaser scan confocal microscope according to a first embodiment of theinvention. Illumination light emitted from a light source 1 which is ofa point light source is formed into parallel light by an illuminationlens 2, only the illumination light having a particular wavelength isselected by and transmitted through a filter 3, and the illuminationlight is reflected by a dichroic mirror 4 which is of light separationmeans. Then, the illumination light is changed to two-dimensional scanlight by a galvano-scanner 5 which is of scanning means, and thetwo-dimensional scan light is collected to an inspection surface of asample 7 by an objective lens 6. The light source 1 and the inspectionsurface of the sample 7 are conjugate to each other in an illuminationoptical system from the illumination lens 2 to the objective lens 6. Theinspection surface of the sample 7 is matched with a focal plane of theobjective lens 6.

Fluorescent light is generated from an irradiation point at which theinspection surface of the sample 7 is irradiated, and the fluorescentlight is collected by the objective lens 6. At this point, due to achromatic aberration of magnification of the objective lens 6, thefluorescent light outgoing from the objective lens 6 travels through anoptical path shifted from that of the irradiation light, and thefluorescent light is substantially changed to non-scan light by thegalvano-scanner 5. The optical path of the fluorescent light is deviatedfrom the optical path of the irradiation light which is of non-scanlight.

The fluorescent light is transmitted through the dichroic mirror 4,lights having unnecessary wavelengths are removed by a filter 8, and thelight is incident to deflection means 9 which is of the two-dimensionaldeflection means. The deflection means 9 is driven in synchronizationwith the galvano-scanner 5 by a computer 10, and the deflection means 9corrects an optical path deviation caused by the chromatic aberration ofmagnification of the objective lens 6. The fluorescent light of whichthe optical path deviation has been corrected forms an image of anirradiation point of the inspection surface of the sample 7 on a pinholeof a pinhole plate 12 by a collective lens 11.

The irradiation point of the inspection surface of the sample 7 isconjugate to the pinhole of the pinhole plate 12 in an image-formationoptical system from the objective lens 6 to the collective lens 11. Thatis, the pinhole plate 12 is provided at a position which is conjugate toa focal plane of the objective lens 6 for the collective lens 11 at awavelength of predetermined illumination light. The light having passedthrough the pinhole is detected by a photodetector 13 and the output istransmitted to the computer 10. The computer 10 forms a two-dimensionalimage from the output of the photodetector 13 to display thetwo-dimensional image on a monitor 14. Thus, the image is obtained.

The optical system of the first embodiment is similar to the opticalsystem of the conventional laser scan confocal microscope except thatthe deflection means 9 is provided. An optical path deviation of thefluorescent light is generated due to the chromatic aberration ofmagnification of the objective lens 6. Accordingly, unless thedeflection means 9 is provided, the image-formation position of thecollective lens 11 differs from the pinhole position of the pinholeplate 12 to generate the limb darkening. The chromatic aberration ofmagnification depends on an angle of view of the objective lens.Accordingly, in the first embodiment, a deflection amount of thedeflection means 9 is determined by working of the computer 10 accordingto a deflection amount of the galvano-scanner, such that wherever theirradiation point is located, the position at which the image of thefluorescent light is formed with the collective lens 11 is matched withthe pinhole position of the pinhole plate 12.

The optical path deviation of the fluorescent light caused by thechromatic aberration of magnification is generated depending on the typeof the objective lens used, the wavelength of the illumination light(excitation wavelength), and the fluorescent light wavelength (peakwavelength of fluorescent light emitted from fluorescent dye) inaddition to a deflection amount of the galvano-scanner 5. Accordingly,the computer 10 performs operations using the type of the objective lensused, the wavelength of the illumination light, the fluorescent lightwavelength, and a deflection amount of the galvano-scanner 5 when adeflection amount of the deflection means 9 is determined for thedeflection amount of the galvano-scanner 5.

Examples of a parameter used to determine the deflection amount of thedeflection means 9 include the type of the objective lens 6, thewavelength of the illumination light (excitation wavelength), thefluorescent light wavelength (peak wavelength of fluorescent lightemitted from fluorescent dye), and an angle (hereinafter referred to asdeflection angle) formed by the illumination light emitted from thegalvano-scanner 5 and the optical axis (the optical axis of theobjective lens). The chromatic aberration of magnification is computedfrom the parameters at each deflection angle of the fluorescent lightwith respect to the illumination light, and the chromatic aberration ofmagnification is converted into the deflection amount (rotation angle ofdeflection means) of the deflection means 9. A pitch of the computeddeflection angles can arbitrarily be selected.

After the objective lens, the illumination light wavelength, and thefluorescent light wavelength are determined, deflection amounts of thedeflection means 9 are determined for the whole region of the deflectionangle before actual images are obtained. The deflection amounts of thedeflection means 9 are stored in a memory of the computer 10.

A first method for determining the deflection amounts of the deflectionmeans will be described below.

FIG. 6 is a view showing a table used to determine the deflectionamounts by the first method. The table of FIG. 6 shows a value of thechromatic aberration of magnification at each deflection angle at eachfluorescent light wavelength when the illumination light has thewavelength of 405 nm and the objective lens is of 10×.

K11-K14, K21-K24, K31-K34, K41-K44 and K51-K54 of FIG. 6 designatevalues of the chromatic aberration of magnification at each fluorescentlight wavelength for the illumination light having the wavelength of 405nm, and K11-K14, K21-K24, K31-K34, K41-K44 and K51-K54 are previouslycomputed from design specifications of the objective lens. Tables of thetype shown in FIG. 6, the number of which corresponds to number of thecombinations of the objective lenses to be mounted and the wavelengthsof the illumination light (excitation wavelengths), are stored in thecomputer 10. The classification of the objective lens is not limited tothe magnification, but preferably the classification is performed ineach model of the objective lens.

Using the table of FIG. 6, a deflection amount of the deflection means 9can be computed at the fluorescent light wavelength of 412 nm and thedeflection angle of 2.5 degrees as below. First the chromatic aberrationof magnification at the fluorescent light wavelength of 412 nm iscomputed at the deflection angle of 2.5 degrees. Linear interpolation isperformed to a series of data of fluorescent light wavelength of 410 nmand a series of data of fluorescent light wavelength of 415 nm tocompute the chromatic aberration of magnification corresponding to thefluorescent light wavelength of 412 nm at each deflection angle. Usingthe computed values of chromatic aberration of magnification, linearinterpolation is performed to chromatic aberration of magnification atthe deflection angle of 2 degrees and chromatic aberration ofmagnification at the deflection angle of 3 degrees to compute thechromatic aberration of magnification at the deflection angle of 2.5degrees. A deflection amount α of the deflection means 9 is expressed byα=tan⁻¹(K/F)  (1)where K designates chromatic aberration of magnification and Fdesignates a focal distance of the objective lens. A deflectiondirection of the deflection means 9 exists in the same plane as thedeflection direction of the galvano-scanner 5, and the deflectiondirection of the deflection means 9 is such a direction as to cancel thechromatic aberration of magnification.

Additionally, a modification of the first method for determining thedeflection amount of the deflection means will be described below. FIG.7 is a view showing a table used to determine a deflection amount by amodification of the first method. The table of FIG. 7 shows a value ofthe chromatic aberration of magnification at each deflection angle ateach wavelength (illumination light and fluorescent light) in areference wavelength when the objective lens is of 10×. S11-S14,S21-S24, S31-S34, S41-S44 and S51-S54 of FIG. 7 designate values of thechromatic aberration of magnification at each wavelength in thereference wavelength, and S11-S14, S21-S24, S31-S34, S41-S44 and S51-S54are previously computed from the design specifications of the objectivelens. The reference wavelength can arbitrarily be selected. A d line(587 nm) can be cited as an example. Tables, the number of whichcorresponds to the number of objective lenses to be mounted, are storedin the computer 10. The classification of the objective lens is notlimited to the magnification, but preferably the classification isperformed in each model of the objective lens.

Using the table of FIG. 7, a deflection amount of the deflection means 9can be computed at the illumination light wavelength of 405 nm, thefluorescent light wavelength of 412 nm, and the deflection angle of 2.5degrees as below. First the chromatic aberration of magnification iscomputed at each deflection angle at the reference wavelengthcorresponding to the illumination light wavelength. That is, linearinterpolation is performed to a series of data of 400 nm and a series ofdata of 410 nm to compute the chromatic aberration of magnification ateach deflection angle at wavelength of 405 nm in the referencewavelength.

Then, using the computed values of chromatic aberration ofmagnification, linear interpolation is performed to the chromaticaberration of magnification at the deflection angle of 2 degrees and thechromatic aberration of magnification at the deflection angle of 3degrees to compute the chromatic aberration of magnification at thedeflection angle of 2.5 degrees at the reference wavelength.

The chromatic aberration of magnification is computed at each deflectionangle at the reference wavelength corresponding to the fluorescent lightwavelength. That is, linear interpolation is performed to the series ofdata of 410 nm and the series of data of 420 nm to compute the chromaticaberration of magnification at each deflection angle at he referencewavelength of 412 nm.

Then, using the computed values of chromatic aberration ofmagnification, linear interpolation is performed to the chromaticaberration of magnification at the deflection angle of 2 degrees and thechromatic aberration of magnification at the deflection angle of 3degrees to compute the chromatic aberration of magnification at thedeflection angle of 2.5 degrees at the reference wavelength of 412 nm.

Finally a difference between the chromatic aberration of magnificationat the deflection angle of 2.5 degrees at the reference wavelength of405 nm and the chromatic aberration of magnification at the deflectionangle of 2.5 degrees at the reference wavelength of 412 nm is computedto obtain the chromatic aberration of magnification at the illuminationlight wavelength of 405 nm, the fluorescent light wavelength of 412 nm,and the deflection angle of 2.5 degrees.

Assuming that K designates the chromatic aberration of magnification andF designates the focal distance of the objective lens 6, the deflectionamount α of the deflection means 9 is computed from the equation (1).The deflection direction of the deflection means 9 exists in the sameplane as the deflection direction of the galvano-scanner 5, and thedeflection direction of the deflection means 9 is such a direction as tocancel the chromatic aberration of magnification.

A second method for determining the deflection amount of the deflectionmeans will be described below.

A good approximation of the chromatic aberration of magnification can becomputed by the following cubic functionK=A×tan³(θ)  (2)where K designates the chromatic aberration of magnification and Adesignates a coefficient determined by a kind of the objective lens anda wavelength.

FIG. 8 is a view showing a table used to determine the deflection amountby the second method. The table of FIG. 8 shows coefficient A of thechromatic aberration of magnification at each deflection angle at eachwavelength (illumination light, fluorescent light) of the referencewavelength when the objective lens is of 10×. A1 to A5 of FIG. 8designate values of the coefficient A of the chromatic aberration ofmagnification at each wavelength of the reference wavelength, and A1 toA5 are previously computed from the design specifications of theobjective lens.

The reference wavelength can arbitrarily be selected. The d line (587nm) can be cited as an example. Tables, the number of which correspondsto the number of objective lenses to be mounted, are stored in thecomputer 10. The classification of the objective lens is not limited tothe magnification, but preferably the classification is performed ineach model of the objective lens.

Using the table of FIG. 8, a deflection amount of the deflection means 9can be computed at the illumination light wavelength of 405 nm, thefluorescent light wavelength of 412 nm, and the deflection angle of 2.5degrees as below. First, using the equation (2), the chromaticaberration of magnification is computed at each deflection angle atwavelengths of 400 nm and 410 nm of the reference wavelength.

Similarly to the modification of the first embodiment, using thecomputed values of chromatic aberration of magnification, the chromaticaberration of magnification is computed at the deflection angles of 2.5degrees at the fluorescent light wavelength of 412 nm with respect tothe illumination light wavelength of 405 nm, and the deflection amount αof the deflection means 9 is computed. The method with the table of FIG.8 has an advantage of reducing the data amount of the table.

A third method for determining the deflection amount of the deflectionmeans will be described below.

In the third method, lens data of the mounted objective lens is storedin the computer 10. The chromatic aberration of magnification of theillumination light with respect to the reference wavelength and thechromatic aberration of magnification of the fluorescent light withrespect to the reference wavelength are computed when the illuminationlight wavelength (excitation wavelength), fluorescent light wavelengthand deflection angle of the illumination light are fed into the computer10. Then the deflection amount of the deflection means 9 is computed ina method like that of the modification of the first method.

The lens data includes a curvature radius of glass constituting theobjective lens, surface separation, and a refractive index (dispersioncoefficient of glass).

Similarly to the modification of the first method, the deflection amountof the deflection means 9 is determined from the computed chromaticaberration of magnification.

The table and the lens data used in the methods can be added when a newobjective lens is added.

The deflection amount determined in the above-described way can bechanged before images are obtained. For example, when all the deflectionamount are changed to zero, the laser scan confocal microscope isoperated in the same manner as the conventional laser scan confocalmicroscope in which the deflection means 9 is not used.

A correction amount of the chromatic aberration of magnification canfinely be corrected by slightly correcting the computed deflectionamount. In such cases, the deflection means 9 is driven according to thecomputed deflection amount to tentatively obtain the image, and a userconfirms a degree of the limb darkening from the image displayed on themonitor 14, thereby slightly correcting the deflection amount. Then theimage is obtained again to confirm the effect of the correction. Theuser repeats the manipulation for correcting and obtaining the imageaccording to the results, whereby the deflection amount of thedeflection means 9 can be determined such that the limb darkening isminimized. At this point, the user corrects deflection amounts only forseveral points of the deflection angles. In other deflection angleranges, deflection amounts are determined by interpolation (linearinterpolation or spline interpolation).

With reference to the correction of the deflection amount of thedeflection means 9, the computer 10 can automatically and sequentiallyrepeat the correction of the deflection amount and the acquisition ofthe image such that the degree of the limb darkening is reduced.

Because the galvano-scanner 5 is formed by the galvano-mirror typescanner, the galvano-mirror type scanner is properly used as thedeflection means 9, such that the galvano-scanner 5 and the deflectionmeans 9 are easily matched with each other. Alternatively, aresonant-mirror type scanner, a scanner in which a mirror is attached toa piezoelectric element, and an acousto-optic element can appropriatelybe used.

FIG. 2 is a view showing an outline of an optical system in a laser scanconfocal microscope according to a second embodiment of the invention.In the following drawings, the same component as that of the firstembodiment is designated by the same numeral, and sometimes thedescription will be omitted. The second embodiment of FIG. 2 is similarto the first embodiment of FIG. 1 except for the following point. Thatis, instead of the deflection means 9 which is of the two-dimensionaldeflection means, deflection means 15 which is of the two-dimensionaldeflection means is provided between the illumination lens 2 and filter3 of the illumination optical system. Alternatively, the deflectionmeans 15 may be provided between the filter 3 and the dichroic mirror 4.Similarly to the first embodiment of FIG. 1, the deflection means 15 isdriven by the computer 10 in synchronization with the galvano-scanner 5.

In the second embodiment of FIG. 2, the deflection means 15 deflects theoptical path of the irradiation light so as to prevent the fluorescentlight emitted from the irradiated position of the sample 7 from failingto pass through the pinhole of the pinhole plate 12 due to the chromaticaberration of magnification of the objective lens 6. The secondembodiment of FIG. 2 is optically equivalent to the first embodiment ofFIG. 1. The deflection amount of the deflection means 15 is determinedin the same way as in the first embodiment.

In the second embodiment, because the galvano-scanner 5 is formed by thegalvano-mirror type scanner, the galvano-mirror type scanner is properlyused as the deflection means 15, such that the galvano-scanner 5 and thedeflection means 15 are easily matched with each other. Alternatively, aresonant-mirror type scanner, a scanner in which a mirror is attached toa piezoelectric element, and an acousto-optic element can appropriatelybe used.

FIG. 3 is a view showing an outline of an optical system in a laser scanconfocal microscope according to a third embodiment of the invention.The third embodiment of FIG. 3 is similar to the first embodiment ofFIG. 1 except for the following point. That is, the deflection means 9which is of the two-dimensional deflection means is eliminated, and apiezoelectric element 16 which two-dimensionally (in directionsperpendicular to optical axis) moves the pinhole plate 12 is providedinstead of the deflection means 9. The piezoelectric element 16 isdriven by the computer 10 in synchronization with the galvano-scanner 5.

In the third embodiment of FIG. 3, the piezoelectric element 16 movesthe position of the pinhole so as to prevent the fluorescent lightemitted from the irradiated position of the sample 7 from failing topass through the pinhole of the pinhole plate 12 due to the chromaticaberration of magnification of the objective lens 6.

In determining a drive amount of the piezoelectric element 16, using oneof the methods described above in determining the deflection amount ofthe deflection means, the chromatic aberration of magnification iscomputed at each deflection angle at the fluorescent light wavelengthwith respect to the illumination light wavelength.

Assuming that K designates the computed chromatic aberration ofmagnification, F designates the focal distance of the objective lens 6,and F2 designates a focal distance of the collective lens 11, a movingamount D of the pinhole plate 12 from a reference position (a positionconjugate to the focal plane of the objective lens set based on thepredetermined illumination light wavelength) is computed by thefollowing equation.D=K×(F2/F)  (3)

Although the pinhole plate 12 is two-dimensionally moved in the thirdembodiment, the same effect can be obtained even if the collective lens11 is two-dimensionally moved in directions perpendicular to the opticalaxis.

FIG. 4 is a view showing an outline of an optical system in a laser scanconfocal microscope according to a fourth embodiment of the invention.The fourth embodiment of FIG. 4 is similar to the first embodiment ofFIG. 1 except for the following point. That is, the deflection means 9which is of the two-dimensional deflection means is eliminated, and twoplane-parallel plates 17 are provided between the light source 1 and theillumination lens 2 instead of the deflection means 9. The twoplane-parallel plates 17 can be rotated about rotation axes 18, 20. Therotation axes 18, 20 are perpendicular to the optical axis, and therotation axes 18, 20 are orthogonal to each other. Rotation of theplane-parallel plates 17 is controlled by the computer 10 insynchronization with the movement of the galvano-scanner 5.

When the plane-parallel plates 17 are rotated, the optical path incidentto the illumination lens 2 is deviated as if the position of the lightsource 1 had been relatively moved. Therefore, the effect equivalent tothat of the second embodiment of FIG. 2 can optically be achieved.Because the plane-parallel plates are inserted immediately after thelight source, advantageously a loss of the light quantity of thefluorescent light can be reduced compared with the plane-parallel platesinserted on the image-formation optical system side.

In determining rotation angles of the two plane-parallel plates 17,using one of the methods described above in determining the deflectionamount of the deflection means, the chromatic aberration ofmagnification is computed at each deflection angle at the fluorescentlight wavelength with respect to the illumination light wavelength.

Assuming that K designates the computed chromatic aberration ofmagnification, F designates the focal distance of the objective lens 6,and F3 designates a focal distance of the illumination lens 2, adeviation amount L between the optical axis and a center axis of theoptical path incident to the illumination lens 2 is computed by thefollowing equation.L=K×(F3/F)  (4)

The two plane-parallel plates 17 are rotated such that the deviationamount of the equation (4) is obtained.

In cases where the loss of the light quantity of the fluorescent lightdoes not become troublesome, the two plane-parallel plates are disposedbetween the collective lens 11 and pinhole plate 12 of theimage-formation optical system. The rotation of the plane-parallelplates is controlled by the computer 10 in synchronization with themovement of the galvano-scanner 5 like the fourth embodiment of FIG. 4,which allows the object of the invention to be achieved.

In such cases, because the galvano-scanner 5 is formed by thegalvano-mirror type scanner, the galvano-mirror type scanner is properlyused as the rotation means, such that the galvano-scanner 5 and therotation-means are easily matched with each other. Alternatively, aresonant type scanner, and a piezoelectric element can appropriately beused.

FIG. 5 is a view showing an outline of an optical system in a laser scanconfocal microscope according to a fifth embodiment of the invention.The fifth embodiment of FIG. 5 differs from the first embodiment of FIG.1 in that a telescope system 19 is provided between the dichroic mirror4 and the deflection means 9, but other optical systems are same.

The deflection amount of the deflection means 9 is extremely small andextremely high accuracy is required to perform the deflection.Therefore, the deviation of the chromatic aberration of magnification ismagnified with the telescope, such that the magnified deviationfacilitates the correction. Accordingly, in the fifth embodiment, thetelescope system 19 (either a Kepler type or a Galileo type) isprovided.

In the embodiments mentioned above, a relationship (a relationship forcollecting the fluorescent light into the pinhole) between a driveamount of the scanning means and a drive amount of one of thetwo-dimensional light deflection means, the means for two-dimensionallyshifting the pinhole plate, the means for two-dimensionally shifting thecollective lens, and the plane-parallel plate is changed when theobjective lens is exchanged. Therefore, desirably the relationships arestored in the computer 10 for each objective lens used, and these meansare driven by the relationship according to the objective lens actuallyused.

In the embodiments, the light emitted from the light source is reflectedand folded by the dichroic mirror 4, the light is incident to thesample, and the generated fluorescent light is transmitted through thedichroic mirror 4. Alternatively, the light emitted from the lightsource may be transmitted through the dichroic mirror 4, and thefluorescent light may be reflected and folded by the dichroic mirror 4.A dichroic prism may be used instead of the dichroic mirror 4.

The filter 3 needs not to be used when the light source 1 is monochrome.The filter 8 may be located at any position between the dichroic mirror4 and the photodetector 13. As the wavelength of the light source isshortened like the UV region and the wavelength of 405 nm, the chromaticaberration of magnification of the objective lens is increased, andtherefore the embodiments of the invention are effectively applied.

Desirably a pupil relay lens and an image-formation lens are disposedbetween the objective lens 6 and the galvano-scanner 5. In such cases,an image of the illumination light two-dimensionally scanned by thegalvano-scanner 5 is tentatively formed by the pupil relay lens, and theillumination light is incident to the image-formation lens to return toparallel light. Then the illumination light is incident to the objectivelens 6 and collected onto the inspection surface of the sample 7. Theobjective lens 6 is changeable.

1. A laser scan confocal microscope comprising: a light source which isdisposed at a position conjugate to a specimen in an illuminationoptical system to emit illumination light; light separation means forseparating the illumination light and observation light, the observationlight being emitted from the specimen; light scanning means for scanningthe illumination light on the specimen; an objective lens which isdisposed between the light scanning means and the specimen to form afocal point on the specimen, the objective lens being one of a pluralityof objective lenses included in the microscope; a collective lens whichcollects the observation light to a position conjugate to the focalpoint of the objective lens; a pinhole plate which is provided in afocal plane of the collective lens; detecting means for detectingobservation light having passed through a pinhole of the pinhole plate;two-dimensional light deflection means which is disposed between thelight separation means and the pinhole plate, to be driven insynchronization with the light scanning means; storage means in whichdata relating to chromatic aberration of magnification is stored for theobjective lens; and operation means for computing, using the data, arelationship between a deflection amount of the light scanning means anda drive amount of the two-dimensional light deflection means.
 2. Theconfocal microscope according to claim 1, wherein the light scanningmeans and the two-dimensional light deflection means are agalvano-mirror type scanner.
 3. The confocal microscope according toclaim 1, wherein the two-dimensional light deflection means corrects anoptical path deviation between the illumination light and theobservation light, the optical path deviation being generated accordingto a deflection amount of the light scanning means.
 4. The laser scanconfocal microscope according to claim 1, comprising correction meansfor correcting the drive amount using scanning information on the lightscanning means and intensity information on the observation light. 5.The laser scan confocal microscope according to claim 1, wherein datarelating to chromatic aberration of magnification of each of theplurality of objective lenses is stored in the storage means.
 6. A laserscan confocal microscope comprising: a light source which is disposed ata position conjugate to a specimen in an illumination optical system toemit illumination light; light separation means for separating theillumination light and observation light, the observation light beingemitted from the specimen; light scanning means for scanning theillumination light on the specimen; an objective lens which is disposedbetween the light scanning means and the specimen to form a focal pointon the specimen, the objective lens being one of a plurality ofobjective lenses included in the microscope; a collective lens whichcollects the observation light to a position conjugate to the focalpoint of the objective lens; a pinhole plate which is provided in afocal plane of the collective lens; detecting means for detectingobservation light having passed through a pinhole of the pinhole plate;two-dimensional light deflection means which is disposed between thelight separation means and the pinhole plate, to be driven insynchronization with the light scanning means; storage means in which arelationship between a deflection amount of the light scanning means anda drive amount of the two-dimensional light deflection means calculatedusing data relating to chromatic aberration of magnification is storedfor the objective lens.
 7. The laser scan confocal microscope accordingto claim 6, wherein the relationship between the deflection amount ofthe light scanning means and the drive amount of the two-dimensionallight deflection means calculated using data relating to chromaticaberration of magnification for each of the plurality of objectivelenses is stored in the storage means.